KR940005583B1 - Method of producing human-lysozym - Google Patents

Abstract

A process for the preparation of human lysozyme and the human lysozyme protein itself are described.

Description

How to prepare human lysozyme

Figure 1 shows the results of primer extension experiments where Ei-ON19 and ON20 are primers and the template is RNA isolated from cell line U-937 (KFCC-10348).

2 shows the results of dot-blot hybridization.

Figure 3a shows the structure of HLZ cDNA (clone HL 14-1),

Figure 3b shows a method for measuring the nucleotide sequence of HLZ cDNA.

Figure 4a shows the DNA sequence of HLZ cDNA of clone HL 14-1,

4b shows a restriction map of HLZ cDNA of clone HL 14-1.

5 is a comparison with the known HLZ amino acid sequence (upper row) and the amino acid sequence derived from the cDNA of clone HL 14-1 (lower row).

Figure 6 shows the multiclonal hybrid vector pEAS102.

Figure 7 shows how the ADHI promoter is linked to the α-factor reader, followed by construction of pWS215D with the HLZ gene linked in the correct orientation to the α-factor reader.

FIG. 8 shows a linker linkage method using a HindIII linker and a linker linkage method using SalI and HindIII linkers to introduce the HLZ gene into the α-factor reader or in the correct orientation with respect to the α-factor promoter or terminator.

Figure 9 shows a method for mutagenesis in vitro using synthetic oligonucleotides EBI 124 and EBI 234 for the exact transition between the α-factor reader and the HLZ gene.

Figure 10 shows how to construct pWS235D with the correct 3 'end of the HLZ gene introduced in the correct orientation in front of the ADHII terminator.

11 shows the distance of the BamH I site (pWS257D) between the HLZ gene and the ADH II terminator, and the method of constructing the expression cassette (pWS290D).

FIG. 12 shows a method of cloning the α-factor gene from the derivative pαF of pUC13 as an EcoR I fragment in vector V2 (a derivative of pUC18) (pWS230D).

Figure 13 illustrates a method of linking the HLZ gene in the correct orientation to the α-factor promoter and terminator (pWS231D).

14A and 14B show expression under the control of the α-factor promoter by linking the distance of the Pst I site nearest to the 5 ′ end of the α-factor gene (pWS294D) and the mutated PstI fragment A2 within a single Pst I site of pWS294D. The method for constructing the cassette pWS296D is shown.

The present invention relates to a method for synthesizing and separating cDNA encoding human lysozyme protein, a method for preparing human lysozyme, and human lysozyme protein itself.

The onset is also described by way of example in the aspects described below, and relates to the possibility of its therapeutic use.

In particular, the present invention relates to bacterial plasmids containing a cDNA sequence for a portion of a human lysozyme signal peptide, a cDNA sequence for a mature mature lysozyme protein, and a non-coding sequence portion of the human lysozyme gene at the 3 'end.

The invention also relates to expression vectors such as plasmids containing nucleotide sequences encoding human lysozyme as inserts, and host organisms or cultures that allow for the preparation of human lysozyme.

Lysozyme is a 1,4-beta-N that cleaves the glycosidic bond between C-1 of N-acetyl-muramic acid (MurNAc) and C-4 of N-acetylglucosamine (GlcNAc) in bacterial faptidoglycans It is defined as acetylmurada.

(1) Alexander Fleming discovered in 1922 that egg whites as well as various tissues and secretions can dissolve some gram-positive bacteria.

He named the solubility factor lysozyme, an enzyme that can dissolve bacteria. Fleming found that lysozyme is present in homogeneous tissues from the cartilage and stomach, tears, saliva, bile, nasal secretions, pathological urine, serum and leukocytes, but not in healthy urine, cerebrospinal fluid or sweat.

(2) Lysozyme activity as an antibacterial agent was based on both its direct bacteriolytic activity and the stimulatory effect associated with the phagocytosis of macrophages. (3, 4)

An important role of lysozyme as a mediator in repelling microorganisms by alveolar macrophages has been described in rats, where phagocytosis damaged by lysozyme occurs quickly while complete bacteria do not have phagocytosis.

(3) Similarly, it has been found that lysozyme can improve the phagocytic activity of polymorphonuclear leukocytes (5) and macrophages (4). Intraoral microorganisms, namely Streptococcus mutans, Veillonella alcalescens and Actinomyces viscous tests were performed. As a result, it has been found that various mechanisms are responsible for bacteriostatic, lytic and bactericidal properties, and that enzymes are not only a selective factor for oral microorganisms but also an effective factor (6). Another obvious action of lysozyme includes immunostimulation (7) and immunological and non-immunological monitoring (8) of the host membrane for oncogenic transformation. Measurement of lysozyme from serum and / or urine is used to diagnose various diseases or as an indicator of disease progression.

Lysozyme serum levels are significantly reduced in acute lymphoblastic leukemia, whereas serum lysozyme concentrations in serum are significantly increased in chronic myeloid leukemia and acute monocytic and osteomyeloid leukemia (9, 10).

Lysozyme is therapeutic in the treatment of various bacterial and viral infections (large and shingles), colitis, various forms of pain, allergies, inflammation and in the pediatric field (converting milk to a form suitable for infants by adding lysozyme). It can be used effectively.

Lysozyme can interact with other biologically active proteins to express their full activity (Adinolfi, In: Lampert, Woods (eds), Academic Press, London, 1981, 19-47). Such ingredients include, for example, sIgA in milk, which enhances the antibacterial activity of lactotransferrin, which inhibits replication of certain microorganisms by forming complements, iron-chelate complexes, and lactotransferrin. It may be an antibody such as (Spik et al., Bull. Eur. Physiopath. Resp. 19, 123-130, 1983). Lysozyme, lactotransferrin and immunoglobulin also coexist in a variety of natural secretions, such as saliva, tears, and various forms of milk (Jorieux et al., Protides of the biological Fluids, Proc. 31st Coll. 1984), bronchial mucosa And egg whites. Lysozyme can additionally be used to help relieve rheumatic fever and rheumatic pain and has therapeutic activity against diseases in the form of rheumatoid or arthritis (Third Int. Symp. On Flemings lysozyme, Milan 1964).

Later, lysozyme was found to also have analgesic properties (Bianchi, Eur. J. Pharmacol. 71, 211-221, 1981). More recently, lysozyme has been shown to have anticitolytic activity (Bianchi, Clin, Exp. Pharmacol. Physiol. 10, 45-52, 1983). The activity of such a wide range of lysozyme accounts for a correspondingly wide range of therapeutic uses, which means that lysozyme is of considerable economic importance.

In all of the pharmaceutical applications of lysorose listed herein, human lysozyme (HLZ) is preferred over lysozyme obtained from egg whites because of the undesirable effects of hypersensitivity and / or allergy when using different species of lysozyme. This is because side effects are more likely to occur and can interfere with treatment.

To date, human milk and human placenta have been the only commercial source for obtaining human lysozyme. But. The availability of these starting materials is very limited and it is clear that different batches of different products will be obtained. Therefore, it is advantageously necessary to produce human lysozyme by microorganisms using widely developed industrial microbiology and recently developed recombinant DNA technology.

Genes for egg white lysozyme were isolated (11,12), and the nucleotide sequence of egg yolk lysozyme mRNA and the exon located with flanking regions on the gene were determined (13).

In addition, the nucleotide sequence of the lysozyme gene of bacteriophage T4 was also identified (14). The amino acid sequence of HLZ is known (21,22,23), but the nucleotide sequence encoding HLZ shown in the present invention is not previously known. EP 181,634 describes the expression of human lysozyme in yeast and Bacillus subtilis. Aside from the fact that no specific yeast terminators and true HLZ genes were used in European Patent Application No. 181,634, this HLZ DNA sequence does not have a preliminary DNA sequence which, when expressed in yeast, encodes a leader fabtide. Therefore, the HLZ formed cannot be transported from the host, and as a result it is difficult to form an accurate tertiary structure by the formation of the corresponding disulfide bonds. Therefore according to EP 181,634 there is no description of how to obtain true HLZ. There is also no explanation how the starting methionine is removed.

The object underlying the present invention relates to a method for synthesizing and cloning a cDNA with minimal information of the amino acid sequence of HLZ and a method for expressing a biologically active HLZ protein using the cDNA.

This problem was solved by revealing that a hybrid hybrid plasmid containing cDNA encoding HLZ can be constructed using mRNA as a template. As part of a bacterial hybrid vector, the HLZ DNA can be derived from human HLZ-producing cells. Examples of suitable cells are white blood cells from placental cells, acute monoblastic and / or osteomyeloid leukemia patients, human colon cancer cells, U-937 cell lines, or other cell lines producing HLZ. In a preferred embodiment of the present invention, U-937 cells (ATCC CRL1593) from Korea (Korean Deposit Institution: Korean spawn association, Accession No .: KFCC-10348, Deposit Date: April 2, 1987) are used.

HLZ DNA can be isolated from the gene bank containing the HLZ gene. In the present invention, chromosomal DNA is undesirable because it usually contains introns in its coding region that cannot be cleaved using yeast (eg, egg white lysozyme gene contains three introns). (13).

In the present invention, human lysozyme cDNA is preferred. MRNA isolated from U-937 cells (KFCC-10348) is a preferred template for human lysozyme cDNA synthesis. To synthesize the first strand of the HLZ cDNA, the reaction is initiated using oligo (dT) ₁₈ , which is preferably paired with the 3 ′ end of the above-mentioned mRNA, as a primer. Single-chain cDNAs are further extended in the presence of dNTPs and reverse transcriptases. Subsequently, single-chain cDNA can be converted into double-stranded cDNA by various well-known methods. In the present invention, the Gubler and Hoffmann methods 15 are preferred. Synthesis of the second chain of cDNA from single-chain cDNA is performed by treating the mRNA hybrid with RNaseH and DNA polymerase I in the presence of dNTPs. Using this method a double stranded cDNA is obtained, which can itself be cloned directly.

Cloning of double-stranded cDNAs in bacterial vectors can be carried out by various known methods. Double-stranded cDNAs can be cloned directly as “smooth end” fragments via synthetic linkers or by a method known as “homopolymer tailing.” When cloning HLZ cDNAs, This is preferred.

In this method, terminal transferase is used to form a single chain at the HLZ DNA 3 'end by adding nucleotides (preferably dGTP) to their "smooth" or "adhesive" ends. The bacterial plasmid is linearized with restriction endonucleases (vector pUC9 is preferred when cloning the HLZ cDNA) and the given complementarity terminal (preferably dCTP) to obtain complementary single chain at the 3 'end. The next step is to link the homopolymer-extended double stranded cDNAs with the corresponding complementary end-added vector under suitable conditions. After recombination occurred, the mixture was used to treat CaCl2-treated E. coli. E. coli cells are transformed and then plated onto selective plates. Clones containing HLZ cDNA can be identified by various known methods. In the present invention, screening using radiolabeled synthetic oligonucleotides is preferred. Using the known amino acid sequence of the HLZ protein, two sets of oligonucleotides each of 17 bases in length are synthesized. Due to the fact that different codons can designate one and the same amino acid, each set is a mixture of different oligonucleotides.

Synthesis of all possible combinations ensures that one of the oligonucleotides present is optimally paired with the HLZ gene. Two pools of each of the 17-mer origonucleotides are used to reduce the likelihood that a "false" positive will be selected.

In screening experiments, DNA of bacterial clones containing cDNA inserts are transferred to a nitrocellulose filter and then an insert containing the HLZ gene is identified using a method known as "colony hybridization".

Nitro cellulose filters are hybridized using radiolabeled pools of human lysozyme gene-specific 17-mer under appropriate conditions. After identifying the corresponding clones, the plasmid DNA of each clone was prepared and the insert was prepared by Maxam and Gilbert's method (chemical method) or Sanger's method (synthesis method using dideoxynucleotides). To be sequenced. By measuring DNA sequences from cDNA inserts from positive clones, one can infer the amino acid sequence of a latent protein. Comparing this sequence with the known HLZ amino acid sequence makes it clear whether the cloned cDNA actually contains the coding region for the HLZ protein.

In general, plasmid vectors containing replicon and regulatory sequences can be used with these hosts. These sequences must be derived from host cells and suitable species.

Vectors typically contain, in addition to the site of replication, recognition sequences that allow for phenotypic selection of transformed cells.

For example, this. E. coli is usually E. coli. Transformation is carried out using pBR322, a plasmid derived from E. coli species (Bolivar, et al, Gene 2,95 (1977)). pBR322 contains ampicillin and tetracycline resistance genes, and thus provides a simple way to identify transformed cells. The pBR322 plasmid or other plasmid contains the promoter itself or must be modified to be used for the expression of its own protein by the microorganism. The most frequently used promoters for the production of recombinant DNA are beta-lactamase (penicillinase) and lactose promoter systems (Chang et al., Nature 275,615 (1978); Itakura et al., Science 198,1056 ( 1977); Goeddel et al., Nature 281,544 (1979)] and tryptophan (trp) promoter systems (Goeddel et al., Nucleic Acids Res. 8,4057 (1980); European Patent Application No. 0036 776). It contains. Although the above mentioned are the most common promoters, promoters of other microorganisms have also been developed and used. Gene sequences for HLZ can be used, for example, under the control of the left promoter (P _L ) of bacteriophage lambda. This promoter is one of the promoters known to be particularly strong and adjustable. This can be regulated by lambda repressors (the genes for these inhibitors are known to be present adjacent to restriction sites).

Mutations of this gene encoding thermostable inhibitors can be included in the vector containing the complete HLZ sequence. When the temperature rises to 42 ° C., the inhibitor is inactivated and the promoter has maximum activity. The total mRNA produced under these conditions will be sufficient to obtain a cell containing approximately 10% of that derived from the P _L promoter in its new synthetic ribonucleic acid. In this way, a clone bank can be prepared in which the functional HLZ sequence is located adjacent to the ribosomal binding site at various intervals from the lambda P _L promoter. These clones are then examined to select the clones with the highest yields.

Expression and translation of the HLZ sequence can be performed under the control of other regulatory systems that can be considered "homologous" to the organism in its untransformed form. Thus, for example, chromatin DNA from lactose-dependent E. coli strains contains lactose or lac operon that enables lactose degradation by direct expression of the enzyme beta-galactosidase.

lac operon modulator is Lee. It can be obtained from bacteriophage lambda plac 5 infectious to E. coli. Phage lac operon can be obtained from the same bacterial species by transduction. Regulatory systems that can be used in the method according to the invention can be derived from plasmid DNA from the same organism. The lac promoter-operator system can be induced by IPTG.

Other promoter-operator systems, or portions thereof, may equally well be used: for example, arabinose operator, collisin E ₁ , operator, galactose operator, alkaline phosphatase operator, trp operator, xylose A operator, tac -Promoters, etc.

Preferably the yeast promoter can be coupled to the HLZ encoded region in a manner that ensures effective expression of HLZ. More particularly, the yeast promoter should be located just before the coding region of mature HLZ containing the translational initiation signal (ATG) inserted at the linking site.

In addition, this HLZ expression modulus can be inserted into various yeast vectors by known yeast transformation methods to produce HLZ expression modulus in yeast.

Yeast cells containing the hybrid plasmid can be cultured in various media to isolate the desired product HLZ therefrom and be purified by known methods. On the other hand, enzyme signal sequences can also be inserted during construction of the HLZ expression modulus. In this case, yeast cells transformed with this kind of plasmid can release HLZ into the culture medium through their cell walls, which can be further purified by recovering the desired product HLZ from the culture medium.

Yeast cells are easy to cultivate, large-scale fermentation conditions have already been established, and yeast is endotoxin free, so enzyme cells are particularly preferred as host organisms for expression. Therefore, in the present invention, eukaryotic microorganisms such as yeast cultures can be preferably used. Although many other species are generally obtained as eukaryotic microorganisms, Saccharomyces cerevisiae is most often used.

For expression in Saccharomyces, plasmid YRp 7 [Stinchcomb. et al., Nature 282,39 (1979); Kingsman et al., Gene 7,141 (1979); Tschumper et al., Gene 10,157 (1980)] and plasmid YEp 13 (Broach et al., Gene 8,121-133 (1979)]. Plasmid YRp 7 contains a TRPI-gene which provides a selection marker for yeast mutants (eg ATCC 44076) that cannot grow in tryptophan free media. TRPI-defects as a hallmark of the yeast host genome allow for efficient detection of transformation when cultured without tryptophan. The situation is similar for the plasmid YEp 13 containing the yeast gene LEU 2 which can be used to supplement the LEU-2 mutants. Suitable promoter sequences for yeast vectors include the 5'- flanking regions of the ADHI gene [Ammerer, G., Method of Enzymology 101,192-201,1983], the 3-phosphoglycerate kinase gene [Hitzeman et al. , J. Biol. Chem. 255, 2073, 1980] or other glycolysis genes (Kawasaki and Fraenkel, BBRC 108, 1107-1112 (1982)), for example enolase, glyceraldehyde-3- Genes for phosphate dehydrogenase, hexokinase, pyruvate tecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglucose isomerase or glucosease. In constructing a suitable expression plasmid, the termination sequences associated with these genes can be used at the 3 'end of the sequence to be expressed in the expression vector to provide polyadenylation and termination of the mRNA.

Other promoters that also have the advantage of transcription controlled by growth conditions include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogena. And promoter regions of genes for enzymes responsible for maltose and galactose metabolism.

Promoters regulated by yeast cross locus (e.g., promoters of genes BARI, MEC1, MEC2, STE2, STE5) can be inserted into a temperature-controlled system using temperature dependent sir mutations. Ph.D. Thesis, University of Oregon, Eugene, Oregon (1979), Herskowitz and Oshima, The Molecular Biology of the Yeast Saccharomyces, Part I, 181-209 (1981), Cold Spring Harbor Laboratory].

These mutations affect the expression of the remaining mating cassettes in the yeast and indirectly affect the mating-dependent promoters. In general, however, any plasmid containing a suitable promoter, original replication and termination sequence for yeast is suitable.

The presence of the complete HLZ cDNA sequence allows the construction of an HLZ gene for expression in other microorganisms such as yeast.

Therefore, for the expression of HLZ in yeast cells according to the present invention, component ADH I promoter-α-factor leader-HLZ gene-ADHII terminator or component α-factor promoter-α-factor reader-HLZ gene-α-factor A secretory plasmid consisting of any one of the terminators can be used.

DNA sequences of the ADH I promoter, ADHII terminator and α-factor genes are known. Jeffery, L., et al., J. Biol. Chem. 257,3018-25,1982; Russell, DW et al. , J. Biol. Chem. 258, 2674-82,1983; Nucleic Acids Res. 12,4049-63,1983].

The HLZ gene produced in this manner can be operably linked to a selected yeast promoter. Expression units of this kind can be incorporated into a variety of yeast vectors. They can transform yeast, allowing them to synthesize human lysozyme.

For the expression of HLZ under selective control of the ADH I promoter, a plasmid containing a full α-factor leader following the 3 'end of the ADH I promoter can be constructed. The DNA sequence encoding the mature HLZ (HLZ gene) is linked to the 3 'end of the α-factor leader in the correct orientation towards the α-factor leader. Construction methods in which the correct N-terminus is generated upon expression of the HLZ gene are preferred. For the final construction of the expression cassette, uniform termination of the HLZ gene is performed and the ADHII terminator sequence is imported so that it is located immediately adjacent to the stop codon of the HLZ gene, contributing to the stability of the mRNA. Expression plasmids of this kind contain all of the components described, in the correct order and orientation with respect to each other, and in the correct base potentials between the components ADH I promoter, α-factor reader, HLZ gene and ADHII terminator.

Between the α-factor leader and the next HLZ gene, a protease cleavage site that matures the α-factor is located after the LYS ARG just before the sequence for the first amino acid of the mature HLZ and formed by the host cell. It is particularly advantageous to link so that the correct N-terminus of the protein is obtained.

Expression cassettes of this kind can be linked into various yeast expression plasmids. This is accomplished by the multiplication plasmid YEp13 [Broach, J.R. et al., Gene 8,121-133,1979], pJDB207 [deposited on Dec. 28, 1984 with accession number DSM 3181], YIp5 (Struhl, K, et al., Proc. Natl. Acad. Sci, USA 76,1035-1039,1979 and pEAS102. Vector pEAS102 can be obtained by partially digesting YIp5 with Pst I, completely digesting YIp5 with BamH I and then linking the isolated 4.3 kb fragment (containing URA3 gene) with the 4.4 kb BamH I / Pst I fragment of pJDB207. Can be.

Using these yeast vectors containing this kind of expression cassette, yeast cells of both hybrid types can be transformed by known methods.

The HLZ produced by these transformants and released into the culture medium can be easily separated by known protein purification methods.

Equally preferred is HLZ gene expression under the control of the α-factor promoter. To accomplish this task, the DNA sequence encoding the mature HLZ is linked in the correct orientation between the α-factor promoter and the α-factor reader.

To construct a complete expression cassette, the α-factor promoter, α-factor reader, HLZ gene and α-factor terminator are arranged in sequence.

In this case too, the same base potential occurs between the 3 'end of the α-factor reader and the 5' end of the HLZ gene, as described for the preparation of expression cassettes under ADH I promoter control. An expression cassette of this kind can be introduced into the yeast expression plasmid described above and then used to transform yeast cells by known methods.

Preferred hosts in this case are yeast strains of cross-type α. Cultivation of such transformants and separation of HLZ released by them are carried out by known methods.

Their construction offers a number of advantages, including:

1. Uniform termination of mRNA resulting in increased mRNA stability.

2. Ease of separation of HLZ from supernatant of culture medium.

3. High percentage of HLZ with correct tertiary structure due to the easier formation of disulfide bonds in secreted proteins than in yeast cells.

4. During the secretion period, the signal or leader fabtide component is accurately separated internally from mature HLZ.

In this way, the N-terminus of the correctly defined protein is obtained.

The N-terminus of mature HLZ protein is lysine. The plasmid construct can be chosen such that the protease cleavage site is located before the lysine codon in the secretion plasmid. Thus, the first amino acid of secreted HLZ is lysine. In intracellular production, the starting ATG (methionine) should be placed before the first amino acid of mature lysozyme so that any translation can begin. Unless this methionine is removed by the cellular mechanism, this methionine can be very destructive (active, stable, antigenic).

In addition to microorganisms, multicellular organism cultures are also suitable hosts for HLZ expression.

In theory, any of these cultures may be used, whether derived from vertebrate cell cultures or from invertebrate cell cultures. However, because replication of vertebrate cells in cultures (tissue cultures) has become a common practice in recent years, vertebrate cells are of greatest interest. Tissue culture, Academic press, Kurse and Patterson, Editors ( 1973). Examples of useful host cell lines of this kind include VERO- and HeLa-cells, Chinese hamster ovary (CHO) -cells and W138, BHK, COS-7 and MDCK-cell lines. Expression vectors for these cells typically contain a site of replication (if necessary), and a promoter located in front of the gene to be expressed, with the necessary ribosomal binding site, RNA splicing site, polyadenylation site, and transcription termination sequence. Contains together.

For use in mammalian cells, the regulation on expression vectors is often taken from viral material. For example, promoters generally use those derived from Polyoma, Adenovirus 2, and most often, those derived from Simian Virus 40 (SV 40). . Initiation and termination promoters of SV 40 are particularly useful because they are both readily obtainable as fragments from viruses and also contain the viral replication site of SV 40. See Fiers et al., Nature 273,113 (1978). ]. Smaller or larger fragments of SV 40 can be used as long as they contain a sequence of approximately 250 bp in length from the HindIII cleavage site to the Bgl I cleavage site in their viral replication site. Moreover, promoters or regulatory sequences normally linked to the desired genetic sequences can be used if these regulatory sequences are suitable for the host cell system and are sometimes preferred.

Sites of replication may be provided by corresponding vector constructions to introduce sites from exogenous sites, eg, SV 40 or other virus sources (eg, polyomas, adeoviruses, VSV, PBV, etc.) or host It may be provided by the chromosomal replication mechanism of the cell. If the vector is introduced into a host cell chromosome, the latter method is usually sufficient.

Transformation of cells using a vehicle can be accomplished in a number of ways. For example, this transformation can be performed using calcium, in which the cells are washed in magnesium and DNA is added to the cells suspended in calcium or the cells are exposed to coprecipitation of DNA and calcium phosphate.

In subsequent gene expression, the cells are transferred to medium for selecting transformed cells.

The polyfabtide according to the invention may be any variant of this polyfabtide as well as the mature human lysozyme (HLZ) described in detail.

These modifications include, for example, methods for shortening molecules at the N or C-terminus and replacing amino acids with other groups without substantially affecting activity.

The present invention particularly relates to variants which are readily and commonly obtained by mutation, digestion, translocation or addition as well as the gene sequences encoding the aforementioned HLZ. Any sequence that encodes the HLZ described above (ie, has a corresponding known biologically active spectrum) and is denatured in comparison to the known ones is also included; those skilled in the art will recognize the denatured DNA sequence of the coding region. It can be measured. Similarly, encoding a polyfabtide having an active spectrum of HLZ and using a known sequence (or a portion thereof) under stringent conditions (e.g., at least 85% homologs and preferably at least 90% homologs) By any hybridization.

The substances according to the invention can be formulated in a known manner to produce pharmaceutically useful compositions, that is to say that the polyfabtide according to the invention can be used on its own or in various antibiotics (tetracycline, bacitra New), enzymes (alpha-amylase, papain) or other ingredients such as vitamins.

The following examples illustrate the invention in detail without restricting it.

Abbreviations used in the examples are as follows.

HLZ: human body-lysozyme

RPMI: Rosewell Park Memorial Institute

Tris HCL: Tris- (hydroxy methyl) -aminomethane adjusted pH using HCL

EDTA: Ethylenediaminetetraacetic acid

DEP: diethylpyrocarbonate

SDS: Sodium Dodecyl Sulfate

NaAc: Sodium Acetate

BSA: Bovine Serum Albumin

DTT: 1,4-dithiothritol (1,4-dimercapto-2,3-butanediol)

PCI: Phenol: Chloromform: Isoamyl alcohol (15: 24: 1)

Beta-NAD: Beta-nicotinamide adenine dinucleotide

sscDNA: Japanese cDNA

E. coli: Escherichia coli

SSC: A solution of 0.015 M sodium citrate adjusted to pH 7 using 0.15 M Nacl, 10 N NaOH.

Denhardt's Solution: 0.1% Picol, 0.1% Polyvinylpyrrolidone, 0.1% BSA

TBE: solution of 0.082 M Tris, 0.082 M boric acid, 0.002 M EDTA (pH 8).

Example 1

Analysis of HLZ Activity in Cell Line U-937 (KFCC-10348)

It has been reported that human hematopoietic cell line U-937 produces human lysozyme (16). To use the U-937 cell line as a source of poly A ⁺ RNA for the synthesis of HLZ cDNA, the activity of HLZ released into the medium by U-937 cells is analyzed. RPMI 1640 Batch Containing 10% Calf Fetal Serum, Penicillin (100 Units / ml) and Streptomycin (50 Units / ml) Freshly inoculated U-937 cell line (KFCC-10348) cultures were densely 3X10 ⁵ cells / ml Grow at 37 ° C. for 3 days until Cells are removed by centrifugation and the activity of HLZ released in the medium is analyzed by Gold and Schweiger method (17). After 2 hours of incubation with the radioactive substrate, the amount of released radioactivity is measured in 100 μl aliquots taken from 2 ml of the culture mixture.

Only RPMI medium is used as negative control.

The amount of HLZ secreted by U-937 cells is calculated from the standard curve prepared (activity of human lysozyme [Sigma] from human milk in RPMI medium).

The results are shown in Table 1.

TABLE 1

As shown in Table 1, U-937 cells release about 1.5 μg of HLZ into RPMI medium per ml of medium.

Thus, it can be concluded that U-937 cell line (KFCC-10348) may be a useful source of HLZ mRNA.

Example 2

Preparation of RNA Containing Human Lysozyme mRNA

a) isolation of whole RNA

Human histocytic lymphoma cell line U-937 (KFCC-10348) is grown at 37 ° C. in RPMI 1640 medium containing 10% calf fetal bovine, penicillin (100 units / ml) and streptomycin (50 units / ml) ( 16). Cells were harvested from 1.6 liters of culture by centrifugation, washed once with 50 ml of ice-cold 10 mM Tris HCL pH 7.4 / 140 mM NaCl / 1.5 mM MgCl ₂ , followed by 10 ml of ice-cold 10 mM Tris HCL pH 8.4 / Suspension in 140 mM NaCl / 1.5 mM MgCl ₂ .

Nonidet detergent Noidet P40 is added to bring the final concentration to 0.5%. The suspension is kept on ice for 5 minutes and then mixed vigorously for 10 seconds to dissolve the cell membrane. The nuclei are then removed by centrifugation at 4 ° C. To 10 ml of the supernatant, the following solution is added: 0.5 ml of 20% SDS, 0..25 ml of 2M Tris HCL pH 9.10, 0.1 ml of 500 mM EDTA pH7.5 10 mM Tris HCL equilibrated with pH8 / 1 mM EDTA Vertically distilled phenol is added and the sample is vigorously shaken for 10 minutes, followed by centrifugation to separate the phases.

The aqueous phase is extracted twice as described above and finally extracted using the same volume of chloroform. 3M potassium acetate (1/10 volume of aqueous phase) is added followed by 2.5 volumes of ethanol.

The solution is kept at -20 [deg.] C. overnight, then centrifuged to collect the RNA precipitate, 3 ml washed once with 70% ethanol and dried in vacuo. RNA is dissolved in 3 ml of water to precipitate, then collected, dried and redissolved as described above. About 8 mg of RNA is obtained.

b) Isolation of Poly A ⁺ RNA

Poly A ⁺ RNA is isolated from total RNA on oligo (dT) -cellulose (PL Biochemicals, Inc.). 0.5 g of oligo (dT) cellulose is suspended in sterile water and then left at 14 ° C. overnight. The next day, 0.5 ml of oligo (dT) -cellulose was charged into a column (Biorad Plastic, 18 cm, 1.5 cm) washed with DEP-treated sterile water, followed by 3 ml proteinase K (Type VI, Sigma). Wash with solution and leave for 15 minutes at ambient temperature. Proteinase. K is dissolved in 0.1 × fill buffer (1 × fill buffer: 50 mM Tris HCI pH 7.4, 1M NaCl, 1 mM EDTA, 0.2% SDS) to a final concentration of 1 mg / ml. The column is subsequently washed with 5 ml of sterile 0.1 M NaOH, then with sterile water until the pH of the eluent is 8, and finally with 10 ml of 1 × fill buffer.

The total RNA resuspended in 500 μl of sterile water is diluted with 4.5 ml of fill buffer, heated at 65 ° C. (water bath) for 5 minutes and then loaded onto a prepared Olyso (dT) -cellulose column. The eluate is collected, heated again at 65 ° C. for 5 minutes and then refilled on the column.

The column is washed with 7 ml of fill buffer to wash out poly A RNA. 1 ml of fractions are collected and 0D _Z60 of each fraction is measured. Eluate with 5 ml of poly A ⁺ RNA sterile water. Both poly A RNA and poly A ⁺ RNA are deposited from 0.3 M NaAc overnight at −20 ° C. using ethanol.

Example 3

Analysis of the presence of HLZ mRNA

a) oligonucleotides of mixed sequences are synthesized

Two oligonucleotide pools of mixed sequence homologous to HLZ are synthesized by solid phase phosphoetherether method (20). From the NH ₂ -terminus of the human lysozyme protein, it is selected for the construction of 17-mer oligonucleotide mixing probe Ei-ON19 of sequences (21,22,23) of amino acid residues 63-68 (Tyr Trp Cys Asn Asp Gly). The Ei-ON19 probe consists of 16 mixed oligonucleotides as shown below.

From the NH2-terminus of the human lysozyme protein, it is selected for the construction of the 17-mer oligonucleotide mixing probe Ei-ON20 of sequences (21,22,23) of amino acid residues 26-31 (Ala Asn Trp Met Cys Leu).

The Ei-ON20 probe consists of 32 mixed oligonucleotides as can be seen below.

b) Primer extension analysis

MRNA isolated from U-937 cell line (KFCC-10348) is analyzed by primer extension method for the presence of HLZ mRNA. 100 ng of Ei-ON19 or Ei-ON20 was added to 70 mM Tris HCL, pH 7.6, 1 mM MgCl2, 30 μCi (gamma- ³² P) ATP (5000 Ci / mmol, Amersham PB. 218), 100 μg / ml BSA, 10 mM DTT, Label at the 5 'end for 60 minutes at 37 ° C. in a 20 μl reaction mixture containing 1 mM spermidine and 2-4 units of polynucleotide kinase (New England BioLabs). The reaction is stopped by heating the mixture at 70 ° C. for 10 minutes.

Primer extension reactions were isolated from 50 mM Tris HCL, pH 8.3, 10 mM MgCl ₂ , 10 mM DTT, 4 mM sodium pyrophosphate, 1.25 mM dGTP, 1.25 mM dATP, 1.25 mM dCTP, 1.25 mM dTTP, U-937 cell line (KFCC-10348) MRNA 150ng. The reaction was carried out at 42 ° C. for 60 minutes in a 40 μl volume of reaction solution containing 40 ng of radiolabeled Ei-ON19 or Ei-ON20 and 200 units of reverse transcriptase (BRL). 50 μl of 50 mM EDTA was added to stop the reaction, followed by extraction with PCI and then precipitation with ethanol.

The primer extension was pelleted by centrifugation, dried and then dyed formamide solution consisting of 90% deionized formamide, 10% 10xTRE, 0.1% xylene cyanol FF and 0.1% bromophenol blue Dissolve in 20 μl.

10 μl aliquots from each sample are packed onto 5% acrylamide / 8M ureagel and electrophoresed at 15 watts for 2 hours. The gel is dried and exposed to Kodak X-Omat RP X-ray film in Kodak X-Omatic Cassette C-2. The results of this experiment are shown in FIG.

It is expected that both primers, Ei-ON19 and Ei-ON20, would be complementary to HLZ mRNA and that extension specific products of these primers could be detected using reverse transcriptase on the HLZ mRNA template (24).

Since the length of the HLZ mRNA is unknown, the length of the synthesized DNA cannot be predicted. However, the following has been confirmed: Primer Ei-ON19 binds to HLZ mRNA in the region encoding amino acid residues 63-68. That is, the length of the synthesized DNA is at least 203 dp + X, where X is the number of nucleotides belonging to the leader sequence and the 5 ′ unencoded region. The same calculation for primer Ei-ON20 has a value of 92dp + X.

Therefore, the two bands differ in length by 111dp. From the results shown in FIG. 1, it can be seen that the strongest band synthesized using the primer Ei-ON19 is about 290dp in length and the strongest band obtained using the primer Ei-ON20 is about 175dp. The difference (115dp) between them is so close to the expected value that one can conclude that the analyzed preparation of mRNA isolated from the U-937 cell line (KFCC-10348) contains HLZ mRNA.

Example 4

Construction of the U-937 cDNA Library

a) synthesis of cDNA

The synthesis of single-chain cDNA was carried out at 50 mM Tris HCL pH 8.3, 10 mM MgCl2, 10 mM DTT, 4 mM sodium pyrophosphate, 1.25 mM dGTP, 1.25 mM dATP, 1.25 mM dTTP, 0.5 mM dCTP, 20 μCi (α- ³² P) dCTP ( 3000 Ci / mmol, Amersham, PB. 10205), 40 to 100 μg / ml poly A isolated from 100 μg / ml oligo d (T) ₁₈ (New England BioLabs), U-937 cell line (KFCC-10348). ^{+ In} 50 μl volume of reaction solution containing RNA and 250 units of reverse transcriptase (BRL). The reaction proceeds at 42 ° C. for 60 minutes. 50 μl of 50 mM EDTA is added to stop the reaction and the reaction product is extracted using PCI. RNA: DNA hybrid is precipitated from 2M NH₄ acetate overnight at −20 ° C. using ethanol. The amount of synthesized first chain is determined by analyzing TCA insoluble radioactivity. From a single reaction, 200-300 ng of single-chain cDNA is usually obtained.

To synthesize the second chain, single-chain cDNA is pelleted by centrifugation, washed once with 80% ethanol, dried and dissolved in a suitable volume of distilled water. SscDNA up to 500 ng with 20 ml Tris HCI, pH 7.5, 5 mM MgCl ₂ , 10 mM (NH ₄ ) ₂ SO ₄ , 100 mM KCl, 0.15 mM β-NAD, 50 μg / ml BSA, 40 μM dNTPs, 8 units / ml E. coli SscDNA was treated in 100 μl volume of reaction solution containing RNase H (BRL), 230 units / ml DNA polymerase I (New England BioLabs), and 10 units / ml E. coli DNA ligase (New England BioLabs). It can be converted to single-chain cDNA (dscDNA). The reaction mixture is continuously incubated at 12 ° C. for 60 minutes and at 22 ° C. for 60 minutes. EDTA was added to stop the reaction, bringing the final concentration to 20 mM.

The dscDNA is extracted using PCI and precipitated as described above (15).

b) Cloning of dseDNA

Cloning vectors with low degree of transformation were prepared as follows: 300 μg of pUC9 plasmid DNA was completely cleaved with Pst I restricted endonucleases. The digested plasmid DNA is purified by agarose gel electrophoresis, electroeluted and precipitated using ethanol.

Agarose-gel purified vectors yield about 1 to 2 colonies / ng in transformation experiments, whereas transformation efficiency with supercoiled pUC9 is about 2000 to 4000 colonies per ng of plasmid DNA.

The pUC9 plasmid DNA thus prepared was continuously transferred to 100 mm potassium cacodylate pH 7.5, 2 mM CoCl2, 0.2 mM DTT, 0.9 mM dCTP, 5 μCi of (5- ³ H) dCTP (19 Ci / mmol, Amersham T. 352), Terminal addition is performed in 50 μl volume of reaction solution containing 5 mg / ml BSA, 28 μg of Pst I cleaved pUC9 plasmid DNA and 45 units of terminal transferase (PL-Biochemicals). The reaction mixture is incubated at 22 ° C. for 5 minutes, then 50 μl of 25 mM EDTA is added to stop the reaction and then incubated at 70 ° C. for 15 minutes. Under these conditions, 22 nucleotides (length needed to achieve maximum transformation efficiency) are added to each 3 'end (19).

The terminal addition of dseDNA using dGTP is carried out under similar conditions except for reaction with 30 units of terminal transferase for 30 minutes at 37 ° C. using about 100 ng of dseDNA.

50 μl of 25 mM EDTA was added to stop the reaction and heat-inactivated at 70 ° C. for 15 minutes.

Analytical annealing of dGTP-terminated dscDNA with dCTP-terminated pUC9 vector DNA was performed for 120 minutes at 58 ° C. in 50 μl reaction solution containing 10 mM Tris HCL pH 7.5, 1 mM EDTA and 150 mM NaCl. do. Different ratios of dseDNA: vector DNA are used to find the maximum number of clones obtainable per weight of injected dscDNA.

Transformation of CaCl ₂ -Competent E. coli RPI cells (19) mixes 100 μl of these cells with 50 μl of annealing reaction mussels and then on LB plates containing 50 μg / ml of ampicillin. By smearing. The rate of providing the maximum number of transformants is used for scale-up experiments to prepare the final cDNA library.

50 ng of dscDNA was used to generate about 20,000 independent clones.

Example 5

Screening of U-937 cDNA Libraries for HLZ Clone

a) colony hybridization

cDNA libraries are screened for positive transformants containing fragments or all of the HLZ gene. cDNA libraries are first screened by colony hybridization. Transformants that are considered positive are further screened by dot blot analysis.

Transformants were plated on LB + ampicillin (50 μg / ml). Grow overnight at 37 ° C. The colonies are then transferred to nitrocellulose filters (0.45 μm Schleicher and Schuell). Plates and filters are both labeled to enable identification of positive transformants. Carefully place the filter on a flat plate for 20 minutes at room temperature (RT), lift it up, and place it on a 3MM filter (3MM Chr, Whatman) saturated with 0.5N NaOH / 1.5M NaCl (topmost). Colony). After incubation for 15 minutes, the filter is transferred to a dry 3MM filter to remove excess NaOH and left on a 3MM filter pre-soaked with 1M Tris HCL pH 7 / 1.5M NaCl for 3 minutes. The filter is then soaked in 3 × SSC for 20 seconds, then air dried and baked at 80 ° C. for 2 hours. The filter thus prepared is prewashed overnight with gentle stirring at 65 ° C. in a buffer containing 3 × SSC, 0.1% SDS (Serva) and 10 mM EDTA. The buffer is changed several times. Preliminary washing is considered complete if no visible traces of bacterial colonies are detected on the filter.

Immediately after washing, the filter is prehybridized in a solution containing 6 × SSC, 1 × Denharz's solution, 0.5% SDS, 100 μg / ml denatured calf thymus DNA (Sigma) and 0.05% sodium propphosphate. After 3 hours of incubation at 37 ° C., the filter was treated with 6 × SSC, 1 × Denharz's solution, 20 μg / ml yeast tRNA (Type XX.Sigma, 0.05% sodium pyrophosphate and kinase treated 17-mer Ei-ON19). In solution, hybridize overnight using gamma- ³² P) ATP labeled 17-mer Ei-On19 at 37 ° C. Kinase reactions were described in Example 3. After hybridization, the filter is washed in 6 × SSC, 0.5% sodium pyrophosphate as follows: for 5 minutes at room temperature, for 30 minutes at 37 ° C., for 10 minutes at 47 ° C. and (optionally) for 10 minutes at 53 ° C. Three times, the filter is then air dried and exposed to X-ray films (X-Omat, RP, Kodark) with a Dupont thickening screen at -70 ° C for 24 hours. After the film is developed, 48 colonies, which clearly show a stronger signal than background, are selected for further analysis (dot-blot analysis).

b) dot-blot hybridization

48 colonies are inoculated into 2 ml of a sample of LB containing 30 μg / ml ampicillim. Cultures are grown at 37 ° C. with vigorous stirring overnight. DNA preparations are obtained by the method 25 of Birnboim and Doly. Each DNA preparation is suspended in 67.5 μl of water. 7.5 μl of 4N NaOH is added and incubated for 1 hour in a 65 ° C. water bath. The sample is cooled on ice and then 20 μl of 1.5N HCI and 195 μl of 2 × SSC, respectively, are added. The total volume of each sample is 290 μl. A portion of 145 μl was filtered through a Bio-Dot device (Bio-Rad) to break the DNA into two nitrocellulose filters. Before filtration, the nitrocellulose filter is pre-soaked in 2 × SSC for 20 minutes.

After filtration, the filter is air dried and baked at 80 ° C. for 2 hours.

One filter hybridizes with (gamma- ³² P) ATP labeled Ei-ON19 17-mer and the other filter hybridizes with (gamma- ³² P) ATP labeled Ei-ON20 17mer. Hybridization and washing conditions were described in Example 5a. The filter is exposed on Kodak X-Omat RP X-ray film for 6 hours at -70 ° C. Six clones showing positive hybridization signals are identified and the original transformants are named as follows: pHL2, pHL8, pHL14-1, pHL21 pHL23 and pHL35. The size of the cDNA insert in the selected clones is determined by restriction analysis.

The DNA of the clone obtained by Bin Boim and Dolly was digested using BamH I and HindIII and then electrophoresed at 150 V for 4 hours on 0.8% agarose mini-gel. Plasmids pHL14-1, pHL21 and pHL23 contain 500bp long inserts. The length of the insert of pHL2 and pHL8 is 300 dp.

Example 6

Structure and DNA Sequence of HLZ cDNA (Clone HL14-1)

One of the positive clones designated HL14-1 (Example 5b) contains 5 approximately 500 bp long cDNA inserts and is selected for more detailed analysis. The structure of HLZ cDNA is shown in Figure 3a. 200 μg of pHL14-1 plasmid DNA was completely digested with BamH I or HindIII restriction endonuclease, (gamma- ³² P) labeled at the 5 ′ end with ATP (Example 3b), and subsequently Cleavage using a second endonuclease.

Specifically labeled DNA fragments are isolated by polyacrylamide or agarose gel electrophoresis (Figure 3). The labeled DNA is recovered by electrolysis and sequenced by Maxam and Gilbert's method (26). The upper arrow in Figure 3b indicates the direction and range of sequencing performed by this method, and the asterisk indicates the marker site.

The internal portion of pHL14-1 cDNA is sequenced according to Sanger's method (27). The BamH I-HindIII restriction fragment of pHL14-1 containing HLZ cDNA was partially digested using Mnl I restriction endonucleases and the ends were clenopolymerase according to the method (28) of Maniatis et al. Blunt ended using and then cloned into M13 Mp9 digested with Sma I. All DNA manipulations with M13 as well as the cleavage are performed exactly as described in the Amersham booklet (Amersham, “M13 Cloning and Sequencing handbook”). Restriction fragments cloned and sequenced in this manner are shown in Figure 3b (lower arrow). Only the location of the corresponding restriction sites is shown in clone HL14-1. The presence of recognition sites for the endonucleases Mnl I, MaeIII and Hinf I was confirmed experimentally. The results of DNA sequencing are shown in FIG. An open reading frame is found in the HLZ cDNA sequence on top of nucleotide20.

Nucleotides 62-451 correspond exactly to the amino acid sequence for human lysozyme (FIG. 5), followed by the translational stop codon TAA. Nucleotides 20 to 61, encoding 14 hydrophobic amino acid residues (such as 5 'Ile Val Leu Gly Leu Val Leu Ser Val Thr Val Gln Gly Lys Val, etc.), are responsible for HLZ leader or signal fabtide moieties similar to other secreted proteins. Code (29). The amino acid composition of the splice junction of human pre-lysozyme is very similar to that of egg white lysozyme (Leu Gly Lys Val versus egg white lysozyme for human pre-lysozyme) [Weisman LSet al., J. Biol. Chem. 261, 2309-2313, 1986].

Example 7

Construction of pHL14-23

Longest 3 'end of the HLZ insert (clone HL23, plasmid pHL23) (Example 5b) to create the expression plasmid for making HLZ (clonal HL14-1, plasmid pHL14-1) Combine with The 3 'end of HLZ DNA of pHL23 has the following sequence:

The 5 'end to the Pst I site (Figure 4) corresponds to the DNA sequence of pHL14-1. About 1 μg of pHL23 is digested with Pst I and HindIII at 36 ° C. (50 mM Tris HcL pH 8.0,1 mM MgCl ₂ , 50 mM NaCl) and separated on a 1% agarose gel.

The 260 bp long Pst I / HindIII fragment isolated from agarose gels by Drezen's method (Dretzen, G. et al., Anal, Biochem. 112, 295-298,1981) is the 3 'end of the HLZ structural gene. It contains.

This fragment (about 500ng) is linked to pHL14-1 (about 50ng) after removal of the 190bp Pst I / HindIII fragment from pHL14-1.

The DNA ligation reaction was performed overnight at 14 ° C. in a 20 μl mixture of ligation buffer (66 mM Tris HCL pH 7.6,6.6 mM MgCl ₂ , 10 mM DDT, 1 mM rATP) and 1 unit of T4 DNA ligase and using a ligase mixture. Competent cells of E. coli HB101 are transformed according to known methods [Maniatis, T. et al., Molecular Cloning, Cold Spring Harbor Press, S. 220, 1982].

Example 8

Expression cassette construction under ADH I promoter control

a) binding of the ADH I promoter to the α-factor rider

The starting material used is the pUC18 derivative pES103 containing the ADH I promoter as a 1.5 kb long BamH I / Xho I fragment. Instead of pES103, YEp13 (ATCC 37115) [Korea Deposit Authority: Korean spawn association, Accession No .: KFCC-10347, Deposit Date: April 2, 1987] can be used in the same manner [Ammerer, G., Methods in Enzymology] , 101,192-201,1983]. In addition to the Xho I site, there are also Pst I and HindIII sites, allowing 250 bp HindIII / PstI fragments of the α-factor gene from pαF to be linked between the HindIII and PstI sites of pES103, containing a large amount of α-factor reader. (Figure 7). The sequence of the DNA of the HindIII / Pst I fragment with α-factor reader is known (Singh, A, et al., Nucleic Acid Res. 11 (12), 4049-63,1983).

For this purpose 1 μg of pES103 and 5 μg of pαF were completely digested using HindIII and Pst I at 36 ° C. in 50 mM Tris HCI pH 8.0, 10 mM MgCl 2, 50 mM NaCl, and about 50 ng of pES103 (HindIII / Pst I) and About 500 ng of pαF (HindIII / Pst I) was used to connect (Example 7) as described above.

The resulting plasmid, in which the α-factor reader has the correct orientation for the ADH I promoter, transforms E. coli HB101, is designated pWS250D.

The 25 bp α-factor leader sequence lost at the splice junction with the ADH I promoter was combined with the ATG initiation codon in the phosphoester ester method [Efimov, VA, et al., Nucleic Acid Res. 10,6675-94, 1982, replaced with a 40-mer oligonucleotide. For this purpose 1 μg of pWS205D is completely decomposed using Xho I and Pst I at 36 ° C. in 50 mM Tris HCL, pH8.0, 10 mM MgCl ₂ , 50 mM NaCl.

Oligonucleotides of the following sequence having Xho I and Pst I termini are linked to Xho I-Pst I truncated pWS250D.

That is, the oligonucleotide is dissolved in water at a concentration of 10 pmol / μl, 5 μl of the solutions of the two DNA chains are combined, heated to 65 ° C. for 10 minutes, cooled to ambient temperature and 1 μl of this solution is carried out in ligase buffer. Used for ligase reaction under the conditions described in Example 7. The resulting plasmid pWS209D contains a region of about 1.5 kb in length with an ADH I promoter and a full α-factor reader up to the start of the α-factor coding region.

b) binding of the α-ingiider to the HLZ gene

In pWS209D, the α-factor leader sequence ends at the HindIII site and at pHL14-23 the HLZ gene is bound to the 3 'end by the HindIII site and to the 5' end by the Sal I site, so pHL14-23 is converted to 6 mM Tris HCl pH8.0. , 6 mM MgCl _{2, in} 15 mM NaCl, completely digested with Sal I at 36 ° C. and cleaved ends were cleno polymerase (ligase buffer as described in Example 7, 0.5 μg of cleaved DNA, 100 μMtLR dATP each) , dTTP, dCTP, dGTP, 25 μl total volume in 1 unit of Cleno polymerase, 30 minutes at ambient temperature). Purification of the smoothly terminated Sal I degradation product is carried out in a 1% agarose gel. The linker linkage is then performed using synthetic HindIII linkers (New England BioLabs) (Maniatis, T. et al., Molecular Cloning, Cold Spring Harbor Press, page 316, 1982). The linker is dissolved at a concentration of 1 μl per 1 μg of water.

Kinase treatment of the HindIII linker was performed with 1 μl of 10 × linker linase treatment buffer (0.66 M Tris HCL pH 7.9,10 mM spermidine, 0.1 M MgCl ₂ , 150 mM DTT, 2 mg / ml BSA), 1 μl linker, 6 μl of water. And in a reaction mixture consisting of 2 units of T4 DNA kinase at 37 ° C. for 1 hour.

The ligation reaction with the kinase treated HindIII linker was 0.5 μg / μL treated with Sal I and Klenow polymerase overnight at 14 ° C. in 10 μg of 1 × linker kinase treatment buffer containing 10 units of T4 DAN ligase. This is done using pHL14-23.

E. coli HB101 was transformed using the resulting plasmid pWS207D, with two HindIII sites on both sides of the HLZ gene.

After 1 μg of pWS207D was HindIII digested (50 mM Tris HCL pH 8.0, 10 mM MgCl2, 50 mM NaCl, 36 ° C.), the HLZ gene could be linked after the α-factor leader as a HindIII fragment in HindIII cleaved pWS209D (5 μg). have. After separating and isolating the HindIII fragment in a 1% agarose gel for ligase reaction (see Drechen et al., Supra), about 50 ng of pWS207D and about 500 ng of pWS209D were added to the connection buffer (66 mM Tris HCL pH7). .6,6.6 mM MgCl ₂ , 10 mM DTT, 1 mM rATP) and 1 unit of T4 DNA ligase. The reaction is carried out overnight at 14 ° C. The resulting plasmid, containing the HLZ gene in the correct orientation for the α-factor reader or ADH I promoter, is designated pWS215D.

c) the exact base potential between the α-factor reader and the HLZ gene

In order to generate the correct N-terminus for the HLZ gene, it is essential that the protease cleavage site that matures the α-factor is positioned correctly before the sequence for the first amino acid of mature HLZ. Therefore excess nucleotides derived from the construction of cDNA (about 20 dG-dC base pairs) and nucleotides encoding the true HLZ leader sequence must be removed.

Plasmid pWS215D was completely digested using Pst I (50 mM Tris HCL pH8.0, 10 mM MgCl ₂ , 50 mM NaCl, 36 ° C.) and a 500 bp long fragment containing most of the α-factor reader and the 5 ′ end of the HLZ gene. Is separated from a 1% agarose gel according to the method of Carter, P. et al. (See: Drechen et al., Supra).

It is cloned back in M13 amp18-am4 (Carter, p., Bedoulle, H., Winter, G., Nucleic Acid Res. 13 4431-43,1985) and used to transfect E. coli strain TGl. Switch.

A2, a recombinant phage with the desired orientation, is used as a source of single-stranded DNA for mutagenesis in vitro.

Template DNA is prepared for dideoxy-sequencing (M13 Cloning and sequencing handbook, Amersham International plc. Amersham UK).

20-mer oligonucleotide EBI124 having the desired base potential between the α-factor reader and the HLZ gene, and 17-mer selective oligonucleotide EBI234 to correct amber mutations present in the M13 vector Synthesis by the method [Efimov, VA et al., Nucleic, Acid Res. 10,6675-94,1982] and purified by preparative polyacrylamide gel electrophoresis and then primers for the synthesis of DNA second chain. Use as.

Oligonucleotide mutagenesis is carried out substantially according to the methods described in the literature. Zoller, M.J. And Smith.M. DNA 3,479-488,1984]:

Selected oligonucleotide EBI234 with 150 pmol and

Mutagenized primer EBI124 with 150 pmol was prepared using 4 units of T4 polynucleotide kinase (Nws England BioLabs) for 45 minutes at 37 ° C. in 20 μl of a mixture of 50 mM TrisHCL pH8.0, 10 mM MgCl ₂ , 1 mM rATP, 5 mM DTT. Phosphorylated and then heated at 70 ° C. for 10 minutes.

0.5 pmol template DNA in 10 μl of a mixture of 10 mM Tris HCL pH8.0, 10 mM MgCl2, 50 mM NaCl was heated by heating 7.5 pmol of kinase treated primers to 100 ° C. for 3 minutes and then cooling to ambient temperature over 1 hour. To hybridize. The restored mixture is cooled on ice and the volume is adjusted to 20 μg of a mixture of 10 mM Tris HCL pH8.0, 10 mM MgCl 2, 25 mM NaCl, 0.25 mM dNTP (dATP, dTTP, dCTP, dGTP) and 0.25 mM rATP and the primers 1 unit of large fragment of DNA polymerase I (BioLabs) is extended in the presence of 10 units of T4 DNA ligase (BioLabs).

The reaction is carried out at 14 ° C. for 16 hours.

The cell law is then provided by HB2155 while transfecting (without a suppressor) the competent E. coli HB2154 cells using small aliquots of the mixture, ie 0.1, 0.5 and 10 μl. See Carter P. et al., Nucleic Acid Res. 13,4431-4443,1985.

84 of 222 plaques generated from the transfected DNA are transferred onto one LB-plate medium and grown for 16 hours at 37 ° C. as colonies of infected bacteria. Nitrocellulose blots are prepared and screened using ³² P labeled mutagenic oligonucleotide EBI124.

The nitrocellulose filter was pre-washed in 6x SSPE (0.9M NaCl, 0.06M NaH ₂ PO ₄ , 6mM EDTA) for 5 minutes at room temperature, hybridization buffer (6x SSPE, 1% Sarkosyl [Sigma], 100 μg / ml Pre-hybridized at 37 ° C. for 15 min in randomly cut tRNA) and then 16 h at room temperature (22 ° C.) in hybridization buffer containing 0.4 × 10 ⁶ cpm / ml probe. Probe (γ- ³² P) ATP is used to label the 5 'end: 30 pmol of the mutagenic primer EBI124 is 50 mM Tris HCL pH 7.6, 10 mM Mgcl ₂ , 10 mM DTT, 1 mM spermidine, 100 μg / ml BSA and 10 pmol In 20 μl of a mixture of (γ- ³² P) ATP (5000 Ci / mmol, Amersham), phosphorylation is carried out using 4 units t4 polynucleotide kinase (BioLabs) at 37 ° C. for 45 minutes.

The reaction mixture is chromatographed on Biogel P-6DG (Biorad) in TE buffer (10 mM Tris HCI pH 7.5,1 mM EDTA) to separate unincorporated (γ- ³² P) ATP from ³² P-labeled oligonucleotides.

After hybridization, the filter is washed three times for 5 minutes at room temperature in 6 × SSC (0.9M NaCl, 0.09M Na citrate) and 1 minute for 5 minutes at 37,47,5,50 and 56 ° C. in pre-warmed 6X SSC. Wash and radiograph after each wash. Radiographs after washing at 56 ° C. (T _D + 2 ° C.) show one clone A2 / 25 that hybridizes strongly to mutagenic oligonucleotides. Plaque purification of putative mutant phage, rescreening to identify pure mutant phage, followed by sequencing to identify defects.

Double-stranded DNA from clone A2 / 25 was prepared according to the methods of Yanish-Perron et al. (Yanisch-Perron, C., Vieira, J., and Messing, J., Gene 33,103-119,1985) Cleavage using I (50 mM Tris HCL pH8.0, 10 mM Mgcl ₂ , about 5 μG in 50 mM NaCl, 36 ° C.). A 440 bp Pst I fragment having the desired base potential between the α-factor reader and the mature HLZ gene was isolated from a 1% agarose gel as described above (see Drechen et al., supra) and then subjected to Examples 8e and 9d) are used for the ligation reactions described in more detail.

d) the 3 'end of the ADH II terminator and HLZ gene

The 5㎍ pAS5 the Sph I / Xba I (6mM _{Tris-HCL pH7.4 6mM Mgcl 2, 150mM NaCl} , 1mM DTT, 36 ℃) decomposition by using ADH terminator Ⅱ [see: Beier, DR, Young, ET , Nature 300,724 -728,1982; Russell, DW, et al., J. Biol, Chem. 258, 2674-2682,1983] are isolated from plasmid PaS5 and from a 1% agarose gel as described above.

The DNA sequence of the ADH II terminator is described in Russell et al. (See above).

Degradation of pUC18 (Vieira, J. Messing, J., Gene 19,259-268, 1982: Yannish-Perron et al., Gene 33, 103-119,1985) was performed with Sph I and Xba I under the conditions specified in detail above. To use. Subsequently, about 50 g of the digested vector and about 500 ng of the ADHII terminator (Sph I / Xba I fragment) were added to the reaction mixture in 20 μl of ligation buffer (66 mM Tris HCL pH7.6,6.6 mM Mgcl ₂ , 10 mM DTT, 1 ml rATP), Link using 1 unit of T4 DNA ligase overnight at 14 ° C. The resulting plasmid pWS213D is used to transform E. coli HB101.

The HLZ DNA from pHL14-23 further contains 20 dc-dG base pairs derived from the preparation of cDNA and nucleotides of the unencoded region following the stop codon, thus removing them. For this purpose pHL14-23 is digested using MaeIII (20 mM Tris HCL pH 8.0, 6 mM Mgcl ₂ , 350 mM NaCl, 1 mM DTT, 45 ° C.) and separated (method of Drechen et al., Supra). The resulting 280 bp long MaeIII-fragment was precisely cleaved at the stopcode and contained the 3 'end of the HLZ gene.

Adhesion ends by reacting about 0.5 μg of MaeIII digested DNA with 100 μM dATP, dTTP, dCTP, dGTP and 1 unit of Klenow polymerase at ambient temperature for 25 minutes in a total volume of 25 μg ligase buffer (see above). Is filled with Klenow polymerase.

Approximately 1 μg of pWS213D was digested with Sma I (6 mM Tris HCL pH 8.0,6 mM MgCl ₂ , 20 mM KCL) at 36 ° C., and the blunt-terminated MaeIII fragment of pHL14-23 (about 500 ng) was converted to Sma of pWS213D (about 50 ng). To the I site (conditions as described above).

The resulting plasmid carrying the HLZ gene in the correct orientation for the ADHII terminator is denoted pWS235D. This plasmid contains the 3 'end of the HLZ gene, including the stop codon, followed by the ADHII terminator. Between the HLZ gene and the ADHII terminator there is a BamH I and Xba I cleavage site derived from multiple cloning sites of plasmid pUC18.

The BamH I site is removed because it causes problems in later construction. For this purpose, pWS235D was cleaved with BamH I at 36 ° C. (6 mM Tris HCL pH8.0, 6 mM MgCl ₂ , 150 mM NaCl, 1 mM DTT) and the adhesive ends were filled with Clenow polymerase (see above). ) Sal I linker is introduced under the conditions described above (Example 8b). The resulting plasmid is designated pWS257D and used to transform E. coli HB101. The linking sequence between the 3 'end of the HLZ gene and the 5' end of the ADHII terminator is as follows:

3 ′ terminus of HLZ with stop codon TAA as digested with 1 = MaeIII and filled with Klenow polymerase.

2 = rest of the Sma I cloning site (pWS213D)

3 = open BamH I site filled with Clenow polymerase

4 = Sal I Ringer (New England BioLabs)

The other end of the BamH I site of 5 = 3 (open and filled with Klenow polymerase)

6 = Xba I site and beginning of ADHII terminator

e) construction of expression cassettes;

Plasmid pWS215D (about 1 μg) constructed in Example 8b was digested using Pst I and HindIII (see Example 8a for conditions), and the majority of the complete HLZ gene, and α-factor receptor, was generated at 4.2 kb. Cut using fragments. Under the same conditions, pWS257D was digested using Pst I and HindIII and the resulting fragments linked to the Pst I / HindIII site with the 3 ′ end of the ADHII terminator and HLZ gene. The ligase reaction is as described above (Example 8d).

In the resulting plasmid pWS218D, the 3′-end of the HLZ gene ending with the stop codon and adjacent to the ADHII terminator is located directly at the beginning of the α-factor reader. The exact base potential between the majority of the α-factor reader and the 5 'end of the HLZ gene, and these two components, is still deleted to complete the expression cassette. Therefore, the mutagenized 440 bp long Pst I fragment (about 500 ng) prepared in Example 8c is linked to the Pst I cleavage site of pWS218D (about 50 ng) under the conditions described above. Plasmids with 440 bp long Pst I fragment (5 'end of the HLZ gene and most of the α-factor reader (3' end) in the correct culture for the 3 'end of the ADHI promoter or HLZ gene are designated pWS290D.

The plasmid pWS290D thus contains all of the following components described, with the correct components and orientations to each other, and the exact base potentials between each component: 1450 bp ADH I promoter, 260 bp α-factor reader (protease cleavage site) 390 bp HLZ gene (starts with a lysine codon adjacent to the sequence for mature HLZ protein and ends with a stop codon of the HLZ gene), and a 330 bp ADHII terminator.

f) expression of HLZ in yeast

The expression cassette pWS290D (5 μg) was digested with HindIII and BamH I at 36 ° C. (50 mM Tris HCL pH8.0,10 mM MgCl ₂ , 50 mM NaCl) and a multiplex vector pJDB207 (1 μg) also cleaved with HindIII and BamH I ).

Expression cassette pWS290D (5 ng) and multiple copy vector pJDB207 (1 μg) [Beggs, JD, Gene Cloning in yeast, in: Williamson, R. (Ed.), Genetic engineering, Vol. 2, Academic Press, London 1981 , 175-203: DSM 3181] are digested with HindIII and BamH I at 36 ° C. (50 mM Tris HCL pH8.0,10 mM MgCl ₂ , 50 mM NaCl). The prepared HindIII / BamH I fragment (approximately 500 ng) was thus linked to the cloned multiple replicate plasmid pJDB207 (approximately 50 ng) (using ligation buffer and one unit of T4 DNA ligase under the conditions specified in Example 7 for 20 μl of the mixture). ).

Various heterologous yeast strains of Saccharomyces cerevisiae are transformed with this plasmid 129/29 in the following manner (Beggs, JDNature 275,104-109,1978); 10 ⁷ cells / ml were inoculated into 50 ml of YPD from a preculture grown overnight in YPD (1% Bacterial Yeast Extract, 2% Bactopeptide, 2% Glucose), and then the second generation at 28 ° C. (typically 3 hours). Culture). The preparation is centrifuged at 5000 rpm for 5 minutes and the cells are resuspended in 5 ml of SED (1 M sorbitol, 25 mM EDTA, pH 8.0, 50 mM DTT) and stirred slowly at 30 ° C. for 10 minutes. The formulation is centrifuged at 1600 rpm for 5 minutes, resuspended in 5 ml SEC (1 M sorbitol, 0.1 M Na ₃ citrate, pH 5.8, 10 mM EDTA), then 100 μg is taken and suspended in 2.9 ml water. The formation of spheroplasts is measured at 600 nm. It is then treated with 50 μl of glusulase with gentle stirring at 28 ° C. After several treatments, another 100 μl of the globular form is taken to suspend 2.9 ml of water and E ₆₀₀ is measured. When the absorbance at 600 nm drops to about 30% of the initial value (typically after 20 to 30 minutes) the cells are centrifuged at 1600 rpm (bench centrifugation).

The globular is washed once with 5 ml of CaS (1 M Sorbitol, 10 mM CaCl ₂ , 10 mM Tris HCL pH7.5), again centrifuged slowly and dissolved in 0.5 ml of CaS. Take an aliquot of the globular form, add 1 μg of plasmid DNA (129/29) (if the volume of the DNA solution is larger, this should be adjusted for 1M sorbitol), then leave at ambient temperature for 15 minutes and then 1 ml. PEG (20% w / v PEG 4000, 10 mM CaCl ₂ , 10 mM Tris HCL pH 7.5, filtration sterilization) is added. The mixture is left at ambient temperature for 15 minutes, then centrifuged (bench centrifugation) at 1600 rpm and the pellet is 150 μl of SOS (1 M sorbitol, 33% v / v YPD, 6.5 mM CaCl ₂ , 13.5 μg of sterile amino acid selected for this). Resuspend) and leave at 30 ° C for 20 minutes.

3 ml of supernatant agar (1 M solitol, 2.5% agar, 0.67% BYNB wo aa [botto yeast nitrogen base without amino acid], 2% glucose, 1% 100x amino acid mixture) which was previously cooled to 45 ° C. Gently shake in the middle, then pour onto lower layer agar plates (2% agar, 0.67% BYNB wo aa, 2% glucose, amino acid mixtures such as upper agar) and incubate at 30 ° C. for 2-4 days. The 100% amino acid mixture-leu contains 2 g / l adenine sulfate, arginine, uracil, asparagine, glutamine, histidine, methionine, lysine, tyrosine, phenylalanine, valine, threonine and tryptophan. Transformed yeast strain (Saccharomyces cerevisiae) was incubated at 30 ° C. on sc-leu (0.67% bacterium yeast nitrogen base without amino acid, 5% glucose, 1% 100x amino acid mixture-leu) do.

The table below shows the results of lysozyme expression. The amount of lysozyme is measured with an antibody that specifically binds to lysozyme (Elisa-test).

WS21-1 = a leu2 his3 trp pep4

WS21-3 = α leu2 his3 ura3 pep4

WS21-5 = α leu2 his3 trp1 arg1 pep4

Example 9

Construction of Expression Cassettes Under the Control of the α-Factor Promoter

Published references: Bitter, GA et al., Proc. Natl. Acad. Sci. USA 81,5330-34,1984; Brake, AJet., Pros. Natl. Acad. Sci. USA 81,4642-46 , 1984 describes the insertion of heterologous genes between the HindIII site and the Sal I site of the α-factor gene.

The sequence between the EcoR I site and the Hind site acts as a promoter and leader peptide, and the sequence between the Sal I site and the EcoR I site acts as a transcription terminator.

a) Cloning of α-Factor Genes in a Suitable Vector

The α-factor gene is isolated from the pUC13 derivative pαF (5 μg) as an EcoR I fragment (100 mM Tris HCL pH 7.5,5 mM MgCl ₂ , 50 mM NaCl, 1 mM DTT, 36 ° C.) and cloned into vector V2. The vector V2 is constructed as follows. : pUC18 was cleaved using Sma I and HindIII, the ends were filled with Klenow polymerase as described above and the remaining amount of pUC18 was determined by the resulting vector V2 being 3 of the original 10 clonin sites, ie Reconnect to have only EcoR I. Sst I and Kpn I.

Thus, vector V2 no longer contains the HindIII or Sal I site, so that the only cleavage site for these two enzymes is in the α-factor gene.

Vector V2 (1 μg) is digested with EcoR I and about 500 ng of EcoR I-digested pαF is linked to about 50 ng of EcoR I-digested V2 (ligase reaction as described in Example 7). The resulting plasmid pWS230D, transformed with E. coli HB101, is used for subsequent construction. (Examples 9b, c), the Pst I / BglIII fragment from plasmid pWS230D (Figure 14a) is a promoter of about 900 bp in length. It contains.

The Pst I site following this fragment is about 25 bp within the translated region. About 25 bp of the Pst I site is adjacent to the four HindIII sites, and further above is the DNA region required for transcription of the α-factor gene. The leader sequence of the α-factor begins at about 25 bp to the left of this second Pst I site (ATG) and ends somewhat accurately at the first site of the four HindIII sites.

The length is about 28bp. The terminator is between the Sal I site and the EcoR I site.

b) linking the HLZ gene to the α-factor gene

Restriction cleavage sites HindIII and Sal I on both sides of pHL14-23 must be replaced so that the HLZ gene can be introduced in the correct orientation for the α-factor promoter or terminator.

To do this, approximately 5 μg of pHL14-23 was added to Sal I (6 mM Tris HCL pH8.0, 6 mA MgCl ₂ , 150 mM NaCl, 36 ° C.) or Hind III (50 mM Tris HCL pH 8.0, 10 mM MgCl ₂ , 50 mM NaCl, 36 ° C.). ) And the adhesive ends are each filled with Clenow polymerase (0.5 μg DNA cut in 25 μ reaction volume as in Example 8b) and then the reaction mixture is purified on a 1% agarose gel.

The HindIII linker (New England BioLabs) was linked to the Sal I site (pWS207D) as described in Example 8b), and the Sal I linker (New England BioLabs) was connected to the HindIII site (pWS206D) (in each case a DNA fragment). About 0.5 μg / μl) is used to transform E. coli HB101. The resulting plasmids pWS206D and pWS207D (about 5 μg each) were digested using EcoR I and Pst I (50 mM Tris HCL pH 8.0, 10 mM MgCl ₂ , 50 mM NaCl, 36 ° C.) and the ligase described above (Example 7) Linked together under reaction conditions to give plasmid pWS208D.

Here again a problem arises in which the HLZ gene 3 'end described in Example 8ddp has additional nucleotides after the stop codon.

For this reason, the 3 'end of the HLZ gene is shortened to a stop codon as follows: Plasmid pWS257D (as constructed in Example 8d) contains the Sal I site and the ADHII terminator following the 3' end of the HLZ gene up to the stop codon. This is inappropriate for construction. Plasmids pWS208D and pWS257D were prepared using Sal I and Pst I (50 mM Tris HCL pH 8.0, 10 mM MgCl ₂ , 50 mM NaCl for 1 h at 36 ° C. then increasing NaCl concentration to 150 mM and adding Sal I). Completely decompose and isolate on 1% agarose gel (see Drechen et al., Supra).

Sal I / Pst fragment of pWS208D by linking the Sal I / PstI fragment of pWS257D (about 500 ng) to the Sal I / Pst I site of pWS208D (about 50 ng) as described above by containing the 3 ′ end of the HLZ gene Is replaced by Sal I / Pst I fragment of pWS257D.

The resulting plasmid pWS212D is a HindIII / Sal I fragment containing the complete HLZ gene, while the HLZ gene ends with a stop codon at the 3 'end.

Plasmids pWS212D and pWS230D (Example 9a) were used with HindIII and Sal I (HindIII in 50 mM Tris HCL pH 8.0,10 mM MgCl ₂ , 50 mM NaCl, 36 ° C., 1 hour followed by increasing NaCl concentration to 150 mM and adding Sal I) HindIII / Sal I fragment (about 500 ng) in a vector (about 50 ng) that was completely digested and cleaved from pWS212D and opened with Hind III / Sal I (separated from 1% agarose gel as described above) (Reaction as described in Example 7). In the resulting plasmid pWS231D, the HLZ gene is eventually present in the correct orientation for the α-factor promoter and terminator.

c) Pst I site removal from the α-factor gene

The α-factor gene contains two Pst I sites. One that is not present in the coding region is a major obstacle in subsequent construction and is removed by the following method: 10 μg of pWS230D is removed in 50 mM Tris HCL pH 8.0, 10 mM MgCl ₂ , 50 mM NaCl for 10 minutes at 36 ° C. for 10 minutes. By digesting with Pst I, the plasmid pWS230D (see Example 9a for construction) is partially cut using Pst I. Fragments with exactly one cleavage site (fragments B and C in Figure 14a) are separated from 1% agarose gel. Approximately 1 μg of linearized DNA of pWS230D generated to digest 3 ′ adhesion ends with Mung Bean nuclease was subjected to 13 μl of water and ₂ μl of 10 mM ZnCl ₂ , 1 μl of 4M NaCl, 2 μl of 0.3M acetic acid Nat. PH Dissolve in 4.75 and add 2 μl Mung Bean Nuclease (300 U, Pharmacia). The mixture is left at ambient temperature for 15 minutes and then applied directly to a 1% agarose gel to separate the fragments from the gel (see Drechen et al., Supra). The fragments B and C are then reconnected (conditions as specified in Example 7) to destroy the Pst I site delivered during partial digestion and retain the uncut portion. In this way, the desired Pst I moiety no longer exists and the other part can separate the retained plasmid (C ′). This plasmid (C ′) and pWS231D are completely digested using Pst I and BglII (50 mM Tris HCL pH8.0, 10 mM MgCl ₂ , 50 mM NaCl, 36 ° C.).

Linked C ₁ fragment (850 bp) of C ′ (about 500 ng) to Pst I / Bgl I-digested pWS231D (about 50 ng) (ligase reaction as described in Example 7) to give plasmid pWS294D do. This plasmid pWS294D eventually contains an α-factor gene having a Pst I site in which the 3 'end of the HLZ gene was inserted in the correct orientation.

d) expression cassette construction

Plasmid pWS294D contains the α-factor promoter and the starting portion of the α-factor peptide up to the Pst I cleavage site (about 20 bp leader peptide) and then the 3 ′ end of the HLZ gene from the Pst I site to the stop codon.

The complete expression cassette still lacks the 3 'end of the α-factor reader, the 5' end of the HLZ gene and the exact base translocation between these two components. Plasmid pWS294D (about 1 μg) was completely digested with Pst I (50 mM Tris HCL pH8.0,10 mM MgCl ₂ , 50 mM NaCl 36 ° C.), and mutated 440 bp Pst I fragment from plasmid A2 / 25 (Example 8c) (About 500 ng) is linked to the Pst I site of pWS294D as in Example 8e. The resulting plasmid pWS296D contains the 900 bp promoter sequence of the α-factor gene, the 260 bp α-factor leader (same as the length of the sequence described in Example 8e), and the 390 bp HLZ gene (same as the length of the sequence described in Example 8e) And an expression cassette consisting of a 290 bp α-factor terminator.

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Claims (12)

a) mRNA encoding human lysozyme (HLZ) of the human body from a human cell line, preferably U-937 cell line (Korea Deposit Organization: Korean spawn association, accession number: KFCC-10348, date of deposit: April 2, 1987) Isolating, synthesizing the true DNA sequence thereof, b) inserting the DNA sequence into a plasmid to produce a hybrid plasmid capable of replication and expression in the host cell, and c) the host cell, preferably prokaryotic, eukaryotic or mammalian. Animal cells are transformed with the hybrid plasmids produced above to allow HLZ to be expressed by the host cell, and then d) isolating and purifying the protein HLZ secreted by the host cell to prepare human lysozyme. Way. The method according to claim 1, wherein the DNA sequence is the following sequence (A) or (B), followed by a stop codon.

The method of claim 2, wherein the DNA sequence is inserted between the PstI-cut sites of plasmid pUC9. The method of claim 1 wherein the hybrid plasmid used for transformation is plasmid pHL2, pHL8, pHL14-1, pHL21, pHL23, pHL14-23 or pHL35. 5. The method of claim 4, wherein the DNA sequence encoding HLZ hybridizes with an insert of plasmid pHL14-1 or pHL14-23 under stringent conditions and exhibits at least 85% homogeneity, preferably at least 90% homology. . The method according to any one of claims 1 to 5, wherein the DNA region of the hybrid plasmid encoding HLZ is coupled to a promoter, a leader sequence or a terminator, and a translational start signal is located between the coding region for the HLZ and the promoter. Characterized by the above. The host cell according to any one of claims 1 to 6, which is used for transformation and expression. E. coli, Saccharomyces cerevisiae or mammalian cells. 8. The method of claim 1, wherein the expression vector encodes a mature HLZ or contains the nucleotide sequence described in claim 2. 9. The expression vector according to claim 8, preferably pWS290D or pWS296D is composed of the components of the ADH I promoter α-factor reader, HLZ gene and ADHII terminator, or α-factor promoter, α-factor reader, HLZ gene and α-factor Consisting of components of a taminator, characterized in that said expression vector is able to secrete the mature HLZ synthesized. Two mixed sequence oligonucleotide pools homologous to HLZ were synthesized by the solid phase phosphoester ester method, and the DNA sequence of the oligonucleotide was determined by the amino acid sequence A la A sn T rp M et C y s L eu or T yr T rp C ys A It characterized in that it corresponds to sn A sp G ly, a method for producing a DNA oligonucleotide. By transforming the host cellulite plasmid 129/29 as described in claim 7 into a vector capable of replicating and expressing the transformed host, preferably in a vector as described in claim 4, 5 or 9 To contain a nucleotide sequence as described in the method for producing a transformed host. The method according to any one of claims 1 to 11, wherein the recombinant HLZ protein is identical to the true HLZ, or constitutes a biologically active variant thereof, or contains the following amino acid sequence (I) or (II): .

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